Retroviral vector systems, such as lentiviral vector systems, have been proposed as a delivery system. WO98/17815 relates to the removal of many of the accessory genes. WO99/45126 relates to codon optimisation of the gag-pol sequence as a means of seeking to overcome the Rev/RRE requirement for export and to enhance RNA stability
The hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) and an intron may be functionally equivalent [Huang, Z. M. and Yen, T. S. (1995) Mol. Cell. Biol. 15: 3864-386]). Woodchuck hepatitis virus (WHV), a close relative of HBV, also harbors a PRE (hereinafter referred to as WPRE; see U.S. Pat. No. 6,136,597 and U.S. Pat. No. 6,287,814). Insertion of the WPRE in lentiviral vectors resulted in significant enhancement of expression of reporter genes such as luciferase and green fluorescent protein (GFP) in a variety of cells spanning different species [Zufferey, R. et al, (1999) J. Virol 73: 2886-2892]. Stimulation was irrespective of the cycling status of transduced cells.
The WPRE contains three cis-acting sequences important for its function in enhancing expression levels. However, in addition, it contains a fragment of approximately 180 base pairs (bp), which may comprise the 5′ end of the WHV X protein open reading frame, together with its associated promoter. The full-length X protein has been implicated in tumorigenesis [Flajolet, M. et al, (1998) J. Virol. 72: 6175-6180]. This has been directly demonstrated [U.S. Pat. No. 7,419,829] by administration of EIAV vector to fetal mice in utero: mice transduced with vectors containing the wild type EIAV developed liver tumors whereas mice transduced with vectors lacking the WPRE did not develop liver tumors. The tumorigenic activity of Cis-activation of myc family oncogenes due to the insertion of viral DNA into the host genome is known to be a key mechanism of WHV-mediated carcinogenesis (Buendia, M. A. (1994) In C. Brechot (ed.), Primary liver cancer: etiological and progression factors, p. 211-224; CRC Press, Boca Raton, Fla.; Fourel, G. (1994) In F. Tronche and M. Yaniv (ed.), Liver gene expression, p. 297-343; R. G. Landes Company, Austin, Tex.). Furthermore, in human hepatocellular carcinoma, hepatitis B virus X protein truncated at the C-terminal may be more oncogenic than the full length X protein (Tu H et al. 2001 Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues. Cancer Res 61: 7803-7810).
U.S. Pat. No. 7,419,829 involves the replacement of the wildtype WPRE sequence with a mutant WPRE sequence designed to maintain the enhanced transgene expression without expression of the X-protein. It is believed that there are a limited number of changes that may be made to the WPRE without affecting its ability to enhance transgene expression. However, it is known that retroviral reverse transcriptases have low fidelity (an average single mistake incorporated per 10,000 nucleotides reverse transcribed) The possibility remains therefore for any mutated WPRE to revert to a functional wild-type sequence with tumorigenic potential.
Real et al (Appl Microbiol Biotechnol [2011]91:1581) shows that the removal of WPRE in a lentiviral vector significantly decreases transgene expression, as does Ramezani et al (Molecular Therapy [2000]2(5):458). Johnson et al (Gene Therapy [2013]20:607) showed that in a lentiviral system where the transgene is already optimised for high expression, removal of the WPRE has no significant effect on transgene expression: they are however silent on the replacement of the WPRE with a stuffer sequence. EP1757702 describes a gamma-retroviral vector system which can support high transgene expression in the absence of a WPRE: again this is silent on replacement of the WPRE with a stuffer sequence.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.